Protein kinase inhibitors and uses thereof

ABSTRACT

Described herein are benzisoxazole compounds of formula I:  
                 
 
or a pharmaceutically acceptable derivative or prodrug thereof, wherein A-B is N—O or O—N; Ar is an optionally substituted C 5-10  aryl group; R 1  is hydrogen or an optionally substituted group selected from C 1-10  aliphatic, C 5-10  aryl, C 6-12  aralkyl, C 3-10  heterocyclyl, or C 4-12  heterocyclylalkyl; and T, n, R 2  and R 3  are as described in the specification. These compounds are inhibitors of protein kinases, particularly inhibitors of GSK-3 and JAK mammalian protein kinases. The invention also provides pharmaceutically acceptable compositions comprising the compounds of the invention and methods of utilizing those compounds and compositions in the treatment of various protein kinase mediated disorders.

FIELD OF THE INVENTION

The present invention is in the field of medicinal chemistry and relatesto compounds that are protein kinase inhibitors, compositions comprisingsuch compounds and methods of use. More particularly, the compounds areinhibitors of GSK-3 and JAK and are useful for treating disease states,such as diabetes and Alzheimer's disease, that are alleviated by GSK-3inhibitors, and allergic disorders, autoimmune diseases, and conditionsassociated with organ transplantation that are alleviated by JAKinhibitors.

BACKGROUND OF THE INVENTION

The search for new therapeutic agents has been greatly aided in recentyears by a better understanding of the structure of enzymes and otherbiomolecules associated with target diseases. One important class ofenzymes that has been the subject of extensive study is the proteinkinases.

Protein kinases mediate intracellular signal transduction. They do thisby effecting a phosphoryl transfer from a nucleoside triphosphate to aprotein acceptor that is involved in a signaling pathway. There are anumber of kinases and pathways through which extracellular and otherstimuli cause a variety of cellular responses to occur inside the cell.Examples of such stimuli include environmental and chemical stresssignals (e.g. osmotic shock; heat shock, ultraviolet radiation,bacterial endotoxin, H₂O₂), cytokines (e.g. interleukin-1 (IL-1) andtumor necrosis factor α (TNF-α)), and growth factors (e.g. granulocytemacrophage-colony-stimulating factor (GM-CSF), and fibroblast growthfactor (FGF)). An extracellular stimulus may effect one or more cellularresponses related to cell growth, migration, differentiation, secretionof hormones, activation of transcription factors, muscle contraction,glucose metabolism, control of protein synthesis and regulation of cellcycle.

Many disease states are associated with abnormal cellular responsestriggered by protein kinase-mediated events. These diseases includeautoimmune diseases, inflammatory diseases, metabolic diseases,neurological and neurodegenerative diseases, cancer, cardiovasculardiseases, allergies and asthma, Alzheimer's disease or hormone-relateddiseases. Accordingly, there has been a substantial effort in medicinalchemistry to find protein kinase inhibitors that are effective astherapeutic agents. A challenge has been to find protein kinaseinhibitors that act in a selective manner. Since there are numerousprotein kinases that are involved in a variety of cellular responses,non-selective inhibitors may lead to unwanted side effects.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinasecomprised of α and β isforms that are each encoded by distinct genes[Coghlan et al., Chemistry & Biology, 7, 793-803 (2000); Kim and Kimmel,Curr. Opinion Genetics Dev., 10, 508-514 (2000)]. GSK-3 has beenimplicated in various diseases including diabetes, Alzheimer's disease,CNS disorders such as manic depressive disorder and neurodegenerativediseases, and cardiomyocete hypertrophy [WO 99/65897; WO 00/38675; andHaq et al., J. Cell Biol. (2000) 151, 117]. These diseases may be causedby, or result in, the abnormal operation of certain cell signalingpathways in which GSK-3 plays a role. GSK-3 has been found tophosphorylate and modulate the activity of a number of regulatoryproteins. These include glycogen synthase which is the rate limitingenzyme necessary for glycogen synthesis, the microtubule associatedprotein Tau, the gene transcription factor β-catenin, the translationinitiation factor e1F2B, as well as ATP citrate lyase, axin, heat shockfactor-1, c-Jun, c-Myc, c-Myb, CREB, and CEPBα. These diverse targetsimplicate GSK-3 in many aspects of cellular metabolism, proliferation,differentiation and development.

In a GSK-3 mediated pathway that is relevant for the treatment of typeII diabetes, insulin-induced signaling leads to cellular glucose uptakeand glycogen synthesis. Along this pathway, GSK-3 is a negativeregulator of the insulin-induced signal. Normally, the presence ofinsulin causes inhibition of GSK-3 mediated phosphorylation anddeactivation of glycogen synthase. The inhibition of GSK-3 leads toincreased glycogen synthesis and glucose uptake [Klein et al., PNAS, 93,8455-9 (1996); Cross et al., Biochem. J., 303, 21-26 (1994); Cohen,Biochem. Soc. Trans., 21, 555-567 (1993); Massillon et al., Biochem J.299, 123-128 (1994)]. However, in a diabetic patient where the insulinresponse is impaired, glycogen synthesis and glucose uptake fail toincrease despite the presence of relatively high blood levels ofinsulin. This leads to abnormally high blood levels of glucose withacute and chronic effects that may ultimately result in cardiovasculardisease, renal failure and blindness. In such patients, the normalinsulin-induced inhibition of GSK-3 fails to occur. It has also beenreported that in patients with type II diabetes, GSK-3 is overexpressed[WO 00/38675]. Therapeutic inhibitors of GSK-3 are therefore potentiallyuseful for treating diabetic patients suffering from an impairedresponse to insulin.

GSK-3 activity has also been associated with Alzheimer's disease. Thisdisease is characterized by the well-known β-amyloid peptide and theformation of intracellular neurofibrillary tangles. The neurofibrillarytangles contain hyperphosphorylated Tau protein where Tau isphosphorylated on abnormal sites. GSK-3 has been shown to phosphorylatethese abnormal sites in cell and animal models. Furthermore, inhibitionof GSK-3 has been shown to prevent hyperphosphorylation of Tau in cells[Lovestone et al., Current Biology 4, 1077-86 (1994); Brownlees et al.,Neuroreport 8, 3251-55 (1997)]. Therefore, it is believed that GSK-3activity may promote generation of the neurofibrillary tangles and theprogression of Alzheimer's disease.

Another substrate of GSK-3 is β-catenin which is degradated afterphosphorylation by GSK-3. Reduced levels of β-catenin have been reportedin schizophrenic patients and have also been associated with otherdiseases related to increase in neuronal cell death [Zhong et al.,Nature, 395, 698-702 (1998); Takashima et al., PNAS, 90, 7789-93 (1993);Pei et al., J. Neuropathol. Exp, 56, 70-78 (1997); Smith et al.,Bio-org. Med. Chem. 11, 635-639 (2001)].

Small molecule inhibitors of GSK-3 have recently been reported [WO99/65897 (Chiron) and WO 00/38675 (SmithKline Beecham)].

The Janus kinases (JAK) are a family of tyrosine kinases consisting ofJAK1, JAK2, JAK3 and TYK2. The JAKs play a critical role in cytokinesignaling. The down-stream substrates of the JAK family of kinasesinclude the signal transducer and activator of transcription (STAT)proteins. JAK/STAT signaling has been implicated in the mediation ofmany abnormal immune responses such as allergies, asthma, autoimmunediseases such as transplant rejection, rheumatoid arthritis, amyotrophiclateral sclerosis and multiple sclerosis as well as in solid andhematologic malignancies such as leukemias and lymphomas. Thepharmaceutical intervention in the JAK/STAT pathway has been reviewed[Frank Mol. Med. 5: 432-456 (1999) & Seidel, et al, Oncogene 19:2645-2656 (2000)1.

JAK1, JAK2, and TYK2 are ubiquitously expressed, while JAK3 ispredominantly expressed in hematopoietic cells. JAK3 binds exclusivelyto the common cytokine receptor gamma chain (γ_(c)) and is activated byIL-2, IL-4, IL-7, IL-9, and IL-15. The proliferation and survival ofmurine mast cells induced by IL-4 and IL-9 have, in fact, been shown tobe dependent on JAK3- and γ_(c)-signaling [Suzuki et al, Blood 96:2172-2180 (2000)].

Cross-linking of the high-affinity immunoglobulin (Ig) E receptors ofsensitized mast cells leads to a release of proinflammatory mediators,including a number of vasoactive cytokines resulting in acute allergic,or immediate (type I) hypersensitivity reactions [Gordon et al, Nature346: 274-276 (1990) & Galli, N. Engl. J. Med., 328: 257-265 (1993)]. Acrucial role for JAK3 in IgE receptor-mediated mast cell responses invitro and in vivo has been established (Malaviya, et al, Biochem.Biophys. Res. Commun. 257: 807-813 (1999)). In addition, the preventionof type I hypersensitivity reactions, including anaphylaxis, mediated bymast cell-activation through inhibition of JAK3 has also been reported[Malaviya et al, J. Biol. Chem. 274:27028-27038 (1999)]. Targeting mastcells with JAK3 inhibitors modulated mast cell degranulation in vitroand prevented IgE receptor/antigen-mediated anaphylactic reactions invivo.

A recent study described the successful targeting of JAK3 forimmunosuppression and allograft acceptance. The study demonstrated adose-dependent survival of Buffalo heart allograft in Wistar Furthrecipients upon administration of inhibitors of JAK3 indicating thepossibility of regulating unwanted immune responses in graft versus hostdisease [Kirken, transpl. proc. 33: 3268-3270 (2001)].

IL-4-mediated STAT-phosphorylation has been implicated as the mechanisminvolved in early and late stages of rheumatoid arthritis (RA).Up-regulation of proinflammatory cytokines in RA synovium and synovialfluid is a characteristic of the disease. It has been demostrated thatIL-4-mediated activation of IL-4/STAT pathway is mediated through theJanus Kinases (JAK 1 & 3) and that IL-4-associated JAK kinases areexpressed in the RA synovium [Muller-Ladner, et al, J. Immunol. 164:3894-3901 (2000)].

Familial amyoptrophic lateral sclerosis (FALS) is a fatalneurodegenerative disorder affecting about 10% of ALS patients. Thesurvival rates of FALS mice were increased upon treatment with a JAK3specific inhibitor. This suggested that JAK3 plays a role in FALS[Trieu, et al, Biochem. Biophys. Res. Commun. 267: 22-25 (2000)].

Signal transducer and activator of transcription (STAT) proteins areactivated by, among others, the JAK family kinases. Results from arecent study suggested the possibility of intervention in the JAK/STATsignaling pathway by targeting JAK family kinases with specificinhibitors for the treatment of leukemia [Sudbeck, et al, Clin. CancerRes. 5: 1569-1582 (1999)]. JAK3 specific compounds were shown to inhibitthe clonogenic growth of JAK3-expressing cell lines DAUDI, RAMOS, LCl;19, NALM-6, MOLT-3 and HL-60.

In animal models, TEL/JAK2 fusion proteins have inducedmyeloproliferative disorders and in hematopoietic cell lines,introduction of TEL/JAK2 resulted in activation of STAT1, STAT3, STAT5,and cytokine-independent growth [Schwaller, et al, EMBO J. 17: 5321-5333(1998)].

Inhibition of JAK3 and TYK2 abrogated tyrosine phosphorylation of STAT3,and inhibited cell growth of mycosis fungoides, a form of cutaneous Tcell lymphoma. These results implicated JAK family kinases in theconstitutively activated JAK/STAT pathway that is present in mycosisfungoides [Nielsen, et al, Proc. Nat. Acad. Sci. U.S.A. 94: 6764-6769(1997)]. Similarly, STAT3, STAT5, JAK1 and JAK2 were demonstrated to beconstitutively activated in mouse T cell lymphoma characterizedinitially by LCK over-expression, thus further implicating the JAK/STATpathway in abnormal cell growth [Yu, et al, J. Immunol. 159: 5206-5210(1997)]. In addition, IL-6-mediated STAT3 activation was blocked by aninhibitor of JAK, leading to sensitization of myeloma cells to apoptosis[Catlett-Falcone, et al, Immunity 10:105-115 (1999)].

There is a continued need to find new therapeutic agents to treat humandiseases. Accordingly, there is a great need to develop inhibitors ofGSK-3 and JAK protein kinases that are useful in treating variousdiseases or conditions associated with GSK-3 and JAK activation,particularly given the inadequate treatments currently available for themajority of these disorders.

DESCRIPTION OF THE INVENTION

It has now been found that compounds of this invention andpharmaceutical compositions thereof are effective as protein kinaseinhibitors, particularly as inhibitors of GSK-3 and JAK. These compoundshave the general formula I:

or a pharmaceutically acceptable derivative or prodrug thereof, wherein:

-   A-B is N—O or O—N;-   Ar is an optionally substituted C₅₋₁₀ aryl group;-   T is a C₁₋₄ alkylidene chain wherein one or two methylene units of T    are optionally and independently replaced by O, NR, S, C(O), C(O)NR,    NRC(O)NR, SO₂, SO₂NR, NRSO₂, NRSO₂NR, CO₂, OC(O), NRCO₂, or OC(O)NR;-   n is zero or one;-   R¹ is hydrogen or an optionally substituted group selected from    C₁₋₁₀ aliphatic, C₅₋₁₀ aryl, C₆₋₁₂ aralkyl, C₃₋₁₀ heterocyclyl, or    C₄₋₁₂ heterocyclylalkyl;-   each R² is independently selectea from R, halo, CN, OR, N(R)₂, SR,    C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆ aliphatic), OC(═O)R, SO₂R,    S(═O)R, SO₂NR₂, or NRSO₂(C₁₆ aliphatic);-   each R³ is independently selected from R, halo, CN, OR, N(R)₂, SR,    C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆ aliphatic), OC(═O)R, SO₂R,    S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); and-   each R is independently selected from hydrogen, a C₁₋₈ aliphatic    group, or two R on the same nitrogen are taken together with the    nitrogen to form a 4-8 membered heterocyclic ring having 1-3    heteroatoms selected from nitrogen, oxygen or sulfur.

As used herein, the following definitions shall apply unless otherwiseindicated.

The phrase “optionally substituted” is used interchangeably with thephrase “substituted or unsubstituted.” Unless otherwise indicated, anoptionally substituted group may have a substituent at eachsubstitutable position of the group, and each substitution isindependent of the other.

The term “aliphatic” or “aliphatic group” as used herein means astraight-chain or branched C₁-C₁₀ hydrocarbon chain that is completelysaturated or that contains one or more units of unsaturation, or amonocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic (also referred to herein as “carbocycle” or“cycloalkyl”), that has a single point of attachment to the rest of themolecule wherein any individual ring in said bicyclic ring system has3-7 members. For example, suitable aliphatic groups include substitutedor unsubstituted linear or branched alkyl, alkenyl, or alkynyl groupsand hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

The terms “alkyl”, “alkoxy”, “hydroxyalkyl”, “alkoxyalkyl”, and“alkoxycarbonyl”, used alone or as part of a larger moiety include bothstraight and branched chains containing one to twelve carbon atoms. Theterms “alkenyl” and “alkynyl” used alone or as part of a larger moietyshall include both straight and branched chains containing two to twelvecarbon atoms.

The terms “haloalkyl”, “haloalkenyl” and “haloalkoxy” means alkyl,alkenyl or alkoxy, as the case may be, substituted with one or morehalogen atoms. The term “halogen” means F, Cl, Br, or I.

The term “heteroatom” means nitrogen, oxygen or sulfur and includes anyoxidized form of nitrogen and sulfur, and the quaternized form of anybasic nitrogen. Also, the term “nitrogen” includes a substitutablenitrogen of a heterocyclic ring. As an example, in a saturated orpartially unsaturated ring having 0-3 heteroatoms selected from oxygen,sulfur or nitrogen, the nitrogen may be N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as inN-substituted pyrrolidinyl). It is understood that the compounds of thisinvention are limited to those that can exist in nature as stablechemical compounds.

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation, and includes aryl rings.

The term “aryl”, used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic, bicyclicand tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic and whereineach ring in the system contains 3 to 7 ring members. The term “aryl”may be used interchangeably with the term “aryl ring”. The term “aryl”also refers to heteroaryl ring systems as defined hereinbelow.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic”, as usedherein means non-aromatic, monocyclic, bicyclic, or tricyclic ringsystems having five to fourteen ring members in which one or more ringmembers is a heteroatom, wherein each ring in the system contains 3 to 7ring members.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaryalkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclicand tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”.

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl and heteroarylalkoxy and the like)group may contain one or more substituents. Suitable substituents on theunsaturated carbon atom of an aryl, heteroaryl, aralkyl, orheteroaralkyl group are independently selected from halogen, —R^(o),—OR^(o), —O(CH₂)_(y)R^(o), —SR^(o), 1,2-methylene-dioxy,1,2-ethylenedioxy, phenyl (Ph) optionally substituted with R^(o), —O(Ph)optionally substituted with R^(o), —CH₂(Ph) optionally substituted withR^(o), —CH₂CH₂(Ph) optionally substituted with R^(o), 5-8 memberedheteroaryl optionally substituted with R^(o), 5-8 membered heterocycleoptionally substituted with R^(o), —NO₂, —CN, —N(R^(o))₂, —N(R^(o))(CH₂)_(y)R^(o), —NR^(o)C(O)R^(o), —NR^(o)C(O)N(R^(o))₂, —NR^(o)CO₂R^(o),—NR^(o)NR^(o)C(O)R^(o), —NR^(o)NR^(o)C(O)N(R^(o))₂,—NR^(o)NR^(o)CO₂R^(o), —C(O)C(O)R^(o), —C(O)CH₂C(O)R^(o), —CO₂R^(o),—C(O)R^(o), —C(O)N(R^(o))₂, —OC(O)N(R^(o))₂, —S(O)₂R^(o), —SO₂N(R^(o))₂,—S(O)R^(o), —NR^(o)SO₂N(R^(o))₂, —NR^(o)SO₂R^(o), —C(═S)N(R^(o))₂,—C(═NH)—N(R^(o))₂, or —(CH₂)_(y)NHC(O)R^(o), wherein each R^(o) isindependently selected from hydrogen, optionally substituted Cl₆aliphatic, phenyl, —O(Ph), or —CH₂(Ph), wherein y is 0-6. When R^(o) isa C₁₋₆ aliphatic group or a phenyl ring, it may be substituted with oneor more substituents selected from —NH₂, —NH(Cl₄ aliphatic), —N(C₁₋₄aliphatic)₂, —S(O)(C₁₋₄ aliphatic), —SO₂(C₁₋₄ aliphatic), halogen,—(C₁₋₄ aliphatic), OH, —O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, —CO₂(C₁₋₄aliphatic), —O(halo C₁₋₄ aliphatic), or -halo(C₁₋₄ aliphatic); whereineach C₁₋₄ aliphatic is unsubstituted.

An aliphatic group or a non-aromatic heterocyclic ring may contain oneor more substituents. A saturated carbon of an aliphatic group or of anon-aromatic heterocyclic ring may have one or more substituents.Suitable substituents on the saturated carbon of an aliphatic group orof a non-aromatic heterocyclic ring are selected from those listed abovefor the unsaturated carbon of an aryl or heteroaryl group as well as thefollowing: ═O, ═S, ═NNHR*, ═NN(R*)₂, ═N—, ═NNHC(O)R*, ═NNHCO₂(alkyl),═NNHSO₂(alkyl), or ═NR*, where each R* is independently selected fromhydrogen or an optionally substituted C₁₋₆ aliphatic. When R* is C₁₋₆aliphatic, it may be substituted with one or more substituentsindependently selected from —NH₂, —NH(C₁₋₄ aliphatic), —N(C₁₋₄aliphatic)₂, halogen, C₁₋₄ aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN,CO₂H, CO₂(C₁₋₄ aliphatic), O(halo C₁₋₄ aliphatic), or halo(C₁₋₄aliphatic); wherein each C₁₋₄ aliphatic is unsubstituted.

Substituents on the nitrogen of a non-aromatic heterocyclic ring areselected from —R⁺, —N(R⁺)₂, —C(O)R⁺, —CO₂R⁺, —C(O)C(O)R⁺,—C(O)CH₂C(O)R⁺, —SO₂R⁺, —SO₂N(R⁺)₂, —C(═S)N(R⁺)₂, —C(═NH)—N(R⁺)₂, or—NR⁺SO₂R⁺; wherein each R⁺ is independently selected from hydrogen, anoptionally substituted C₁₋₆ aliphatic, optionally substituted phenyl,optionally substituted —O(Ph), optionally substituted —CH₂(Ph),optionally substituted —CH₂CH₂(Ph), or an unsubstituted 5-6 memberedheteroaryl or heterocyclic ring. When R⁺ is a C₁₋₆ aliphatic group or aphenyl ring, it may be substituted with one or more substituentsselected from —NH₂, —NH(C₁₋₄ aliphatic), —N(C₁₋₄ aliphatic)₂, halogen,C₁₋₄ aliphatic, OH, O(C₁₋₄ aliphatic), NO₂, CN, CO₂H, CO₂(C₁₄aliphatic), O(halo C₁₋₄ aliphatic), or halo (C₁₋₄ aliphatic); whereineach C₁₋₄ aliphatic is unsubstituted.

The term “alkylidene chain” refers to a straight or branched carbonchain that may be fully saturated or have one or more units ofunsaturation and has two points of attachment to the rest of themolecule.

A combination of substituents or variables is permissible only if such acombination results in a stable or chemically feasible compound. Astable compound or chemically feasible compound is one that is notsubstantially altered when kept at a temperature of 40° C. or less, inthe absence of moisture or other chemically reactive conditions, for atleast a week.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

One embodiment of the present invention relates to compounds which are2,1-benzisoxazoles, represented by formula I-A shown below. Anotherembodiment of this invention relates to compounds which are1,2-benzisoxazoles, represented by formula I-B shown below:

wherein Ar, T, n, R¹, and R² are as described above for formula I.

Ar is preferably a substituted or unsubstituted five or six-memberedaromatic ring having zero to two ring heteroatoms selected fromnitrogen, sulfur or oxygen. A more preferred Ar is a substituted orunsubstituted six-membered aromatic ring having zero to two ringnitrogens. Most preferably, Ar group is a substituted or unsubstitutedphenyl ring. Preferably, Ar is substituted with one or more substituentsindependently selected from C₁₋₁₀ aliphatic, C₅₋₁₀ aryl, C₆₋₁₂ aralkyl,C₃₋₁₀ heterocyclyl, C₄₋₁₂ heterocyclylalkyl, halo, CN, OR, N(R)₂, SR,C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆ aliphatic), OC(═O)R, SO₂R,S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic), or two substituents onadjacent positions are optionally taken together with their interveningatoms to form a fused 5-8 membered unsaturated or partially unsaturatedring having zero to two heteroatoms selected from nitrogen, oxygen orsulfur; wherein R is as described above for formula I.

R¹ is preferably hydrogen or an aryl ring, such as a phenyl or pyridylring. Optional substituents on R¹ are independently selected fromhalogen, —R, —OR, —OH, —SH, —SR, protected OH (such as acyloxy), —NO₂,—CN, —NH₂, —NHR, —N(R)₂, —NHCOR, —NHCONHR, —NHCON(R)₂, —NRCOR, —NHCO₂R,—CO₂R, —CO₂H, —COR, —CONHR, —CON(R)₂, —S(O)₂R, —SO₂NH₂, —S(O)R, —SO₂NHR,or —NHS(O)₂R, where R is a C₁₋₆ aliphatic group or a substituted C₁₋₆aliphatic group, preferably having one to three carbons. A particularlypreferred substituent on the C₁₋₆ aliphatic group is —SO₂NH₂.

R² is preferably hydrogen or a C₁₋₄ alkyl group, most preferablyhydrogen.

R³ is preferably hydrogen, halo, O(C₁₋₄ alkyl), or a C₁₋₄ alkyl group.Most preferably R³ is hydrogen. Representative examples of compounds offormula I-A are shown below in Table 1. TABLE 1 Examples of Compounds offormula I-A

No. Structure I-A1 

I-A2 

I-A3 

I-A4 

I-A5 

I-A6 

I-A7 

I-A8 

I-A9 

I-A10

I-A11

I-A12

I-A13

I-A14

I-A15

I-A16

I-A17

I-A18

I-A19

I-A20

I-A21

I-A22

I-A23

I-A24

I-A25

I-A26

I-A27

I-A28

I-A29

I-A30

I-A31

I-A32

I-A33

I-A34

I-A35

I-A36

I-A37

I-A38

I-A39

I-A40

I-A41

I-A42

I-A43

I-A44

I-A45

I-A46

I-A47

I-A48

I-A49

I-A50

I-A51

I-A52

I-A53

I-A54

I-A55

I-A56

I-A57

I-A58

I-A59

I-A60

I-A61

I-A62

I-A63

I-A64

I-A65

I-A66

I-A67

I-A68

I-A69

I-A70

I-A71

I-A72

I-A73

I-A74

I-A75

I-A76

I-A77

I-A78

I-A79

I-A80

I-A81

I-A82

I-A83

I-A84

I-A85

I-A86

I-A87

I-A88

I-A89

The compounds of this invention may be prepared in general by methodsknown to those skilled in the art for analogous compounds, asillustrated by the general scheme below and by the preparative examplesthat follow.

Reagents and conditions: (a) ArCH₂CN, KOH, MeOH, room temperature (rt);(b) formic acid, rt (c) N,N-dimethylformamide dimethyl acetal, CH₃CN,80° C.; (d) N-phenylguanidine-HCl, CH₃CN, reflux.

Scheme I above shows a synthetic route for preparing compounds of thepresent invention. For various Ar groups, the intermediate 3 can beobtained commercially or obtained by known methods as shown in steps (a)and (b) above. See R. B. Davis and L. C. Pizzini, J. Org. Chem., 1960,25, 1884-1888. A Mannich reaction provides intermediate 4, which can betreated with phenylguanidine to give the desired compounds 5. It will beobvious to one skilled in the art that phenylguanidine may be replacedwith other arylguanidines, which are readily available, to provide othercompounds of this invention.

Reagents and conditions: (a) R¹NHC(═NH)NH₂—HCl, CH₃CN, reflux; (b)4-Br—C₆H₄—CH₂CN, KOH, MeOH, room temperature (rt); (c) R⁴B(OH)₂,Pd(PPh₃)₄, Na₂CO₃, dioxane

Scheme II above shows an alternative synthetic route where thepyrimidine ring is constructed before the benzisoxazole ring. Steps (a)and (b) are analogous to the corresponding steps shown above in Scheme Iexcept that they are performed in the opposite order. Step (c)illustrates one of many ways known to those skilled in the art in whichcertain compounds of this invention may be modified to provide furthercompounds of this invention. For example, the bromo substituent ofcompound 8 may be replaced by other groups using standard couplingmethods. R⁴ is preferably an aryl or heteroaryl ring. It will be obviousto one skilled in the art that this scheme may be modified to provideother compounds of this invention.

Reagents and conditions: (a) NaH, DMF/THF 1:1, R⁵C(O)Cl, ambient temp;wherein R¹ is —C(O)R⁵; (b) R⁷NCO, DMSO, ambient temp/80° C.; wherein R¹is —C(O)NHR⁷; (c) [from the p-NO₂-phenyl carbamic esters] R⁷NH₂,DMSO/THF 1:1, 80° C.; wherein R¹ is —C(O)NHR⁷. Alternatively, reagentsand conditions for carbamate formation (not shown): (a) R⁶OC(O)Cl, DMSO,DIPEA, ambient temp; wherein R¹ is —C(O)OR⁶.

Scheme III shows general methods for the preparation of compounds ofFormula I wherein NH—R¹ taken together form an amide (shown in step (a)above), carbamate (not shown) or a urea (shown in steps (a) and (c) orstep (b) above). Acylation of the aminopyrimidine with acid chlorides,chloroformates and isocyanates provides amides, cabamates and ureasrespectively. Alternatively, ureas can be generated by a nucleophilicdisplacement reaction with a primary or secondary amine via thecorresponding p-nitrophenylcarbamate.

Reagents and conditions: a) NHR^(o) ₂, Pd(OAc)₂, P-tBu₃, KOtBu, toluene,90° C.

Scheme IV shows a general method for obtaining compounds 2 (scheme I)wherein the Ar group is substituted with an amine functionality as in2b, and wherein R^(o) is as described above. Compounds of type 2b maythen be taken forward according to Schemes I-III.

The activity of a compound utilized in this invention as an inhibitor ofGSK-3 or JAK kinase may be assayed in vitro, in vivo or in a cell lineaccording to methods known in the art. In vitro assays include assaysthat determine inhibition of either the phosphorylation activity orATPase activity of activated GSK-3 or JAK. Alternate in vitro assaysquantitate the ability of the inhibitor to bind to GSK-3 or JAK.Inhibitor binding may be measured by radiolabelling the inhibitor priorto binding, isolating the inhibitor/GSK-3 or inhibitor/JAK complex anddetermining the amount of radiolabel bound. Alternatively, inhibitorbinding may be determined by running a competition experiment where newinhibitors are incubated with GSK-3 or JAK bound to known radioligands.Detailed conditions for assaying a compound utilized in this inventionas an inhibitor of GSK-3 or JAK kinase are set forth in the Examplesbelow.

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. The amount of compound in the compositions of this inventionis such that is effective to detectably inhibit a protein kinase,particularly GSK-3 or JAK kinase, in a biological sample or in apatient. Preferably the composition of this invention is formulated foradministration to a patient in need of such composition. Mostpreferably, the composition of this invention is formulated for oraladministration to a patient.

The term “patient”, as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

The term “detectably inhibit”, as used herein means a measurable changein GSK-3 or JAK activity between a sample comprising said compositionand a GSK-3 or JAK kinase and an equivalent sample comprising GSK-3 orJAK kinase in the absence of said composition.

As used herein, the term “JAK” is used interchangeably with the terms“JAK kinase” and “a JAK family kinase”. Preferably JAK refers to JAK3kinase.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, salt of an ester or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound of this inventionor an inhibitorily active metabolite or residue thereof. As used herein,the term “inhibitorily active metabolite or residue thereof” means thata metabolite or residue thereof is also an inhibitor of a GSK-3 or JAKfamily kinase.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN⁺(C₁₋₄ alkyl)₄ salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the pharmaceuticallyacceptable compositions may be formulated in an ointment such aspetrolatum.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, the pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

The amount of the compounds of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. Preferably, the compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe inhibitor can be administered to a patient receiving thesecompositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Depending upon the particular condition, or disease, to be treated orprevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may also be present inthe compositions of this invention. As used herein, additionaltherapeutic agents that are normally administered to treat or prevent aparticular disease, or condition, are known as “appropriate for thedisease, or condition, being treated”.

For example, chemotherapeutic agents or other anti-proliferative agentsmay be combined with the compounds of this invention to treatproliferative diseases and cancer. Examples of known chemotherapeuticagents include, but are not limited to, Gleevec™, adriamycin,dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan,taxol, interferons, and platinum derivatives.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation: treatments for Alzheimer'sDisease such as Aricept® and Excelon®; treatments for Parkinson'sDisease such as L-DOPA/carbidopa, entacapone, ropinrole, pramipexole,bromocriptine, pergolide, trihexephendyl, and amantadine; agents fortreating Multiple Sclerosis (MS) such as beta interferon (e.g., Avonex®and Rebif®), Copaxone®, and mitoxantrone; treatments for asthma such asalbuterol and Singulair®; agents for treating schizophrenia such aszyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agentssuch as corticosteroids, TNF blockers, IL-1 RA, azathioprine,cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathioprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonianagents; agents for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, and statins; agents for treating liver disease such ascorticosteroids, cholestyramine, interferons, and anti-viral agents;agents for treating blood disorders such as corticosteroids,anti-leukemic agents, and growth factors; and agents for treatingimmunodeficiency disorders such as gamma globulin.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% toabout 100% of the amount normally present in a composition comprisingthat agent as the only therapeutically active agent.

According to another embodiment, the invention relates to a method ofinhibiting GSK-3 or JAK kinase activity in a biological samplecomprising the step of contacting said biological sample with a compoundof this invention, or a composition comprising said compound.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of GSK-3 or JAK kinase activity in a biological sample isuseful for a variety of purposes that are known to one of skill in theart. Examples of such purposes include, but are not limited to, bloodtransfusion, organ-transplantation, biological specimen storage, andbiological assays.

According to another embodiment, the invention provides a method fortreating or lessening the severity of a GSK-3-mediated disease orcondition in a patient comprising the step of administering to saidpatient a composition according to the present invention.

The term “GSK-3-mediated condition”, as used herein means any disease orother deleterious condition in which GSK-3, is known to play a role.Such diseases or conditions include, without limitation, diabetes,Alzheimer's disease, Huntington's, Parkinson's, AIDS associateddementia, amyotrophic lateral sclerosis (AML), multiple sclerosis (MS),schizophrenia, cardiomycete hypertrophy, ischemia/reperfusionandbaldness.

According to another embodiment, the invention provides a method fortreating or lessening the severity of a JAK-mediated disease orcondition in a patient comprising the step of administering to saidpatient a composition according to the present invention.

The term “JAK-mediated disease”, as used herein means any disease orother deleterious condition in which a JAK family kinase, in particularJAK3, is known to play a role. Such conditions include, withoutlimitation, immune responses such as allergic or type I hypersensitivityreactions, asthma, autoimmune diseases such as transplant rejection,graft versus host disease, rheumatoid arthritis, amyotrophic lateralsclerosis, and multiple sclerosis, neurodegenerative disorders such asFamilial amyotrophic lateral sclerosis (FALS), as well as in solid andhematologic malignancies such as leukemias and lymphomas.

In an alternate embodiment, the methods of this invention that utilizecompositions that do not contain an additional therapeutic agent,comprise the additional step of separately administering to said patientan additional therapeutic agent. When these additional therapeuticagents are administered separately they may be administered to thepatient prior to, sequentially with or following administration of thecompositions of this invention.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating an implantable medical device, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Vascular stents, forexample, have been used to overcome restenosis (re-narrowing of thevessel wall after injury). However, patients using stents or otherimplantable devices risk clot formation or platelet activation. Theseunwanted effects may be prevented or mitigated by pre-coating the devicewith a pharmaceutically acceptable composition comprising a kinaseinhibitor. Suitable coatings and the general preparation of coatedimplantable devices are described in U.S. Pat. Nos. 6,099,562;5,886,026; and 5,304,121. The coatings are typically biocompatiblepolymeric materials such as a hydrogel polymer, polymethyldisiloxane,polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinylacetate, and mixtures thereof. The coatings may optionally be furthercovered by a suitable topcoat of fluorosilicone, polysaccarides,polyethylene glycol, phospholipids or combinations thereof to impartcontrolled release characteristics in the composition. Implantabledevices coated with a compound of this invention are another embodimentof the present invention.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

SYNTHETIC EXAMPLES Example 1 N-phenylguanidine

Aniline (30 mmol, 1 equiv.), cyanamide (1.3 g, 31 mmol, 1.03 equiv.),and 4N hydrogen chloride in dioxane (8 mL, 32 mmol) was stirred for 10minutes at room temperature and heated to 80° C. for 18 hours. Themixture was diluted with water (30 mL) and diethyl ether (50 mL). Theaqueous layer was washed with ether (30 mL) and the organic layers werediscarded. The aqueous layer was neutralized with 6N aqueous HCl (6 mL)and diluted with ethyl acetate (50 mL). The aqueous layer was extractedwith ethyl acetate (50 mL) four times. The combined organic layers wereconcentrated under reduced pressure to afford a solid compound. Thesolid was washed with diethyl ether (30 mL) to provide pale yellow titlecompound. The compound was characterized by LC/MS and HPLC.

The following arylguanidine intermediates were prepared by the proceduredescribed above in Example 1 except the aniline was replaced with theappropriate arylamine: N-(4-fluoro-phenyl)-guanidine;N-(6-chloro-pyridin-3-yl)-guanidine; N-(3-chloro-phenyl)-guanidine;N-(3-methoxy-phenyl)-guanidine; N-(3-benzyloxy-phenyl)-guanidine;4-guanidino-benzenesulfonamide; 3-guanidino-benzenesulfonamide.

The following synthetic intermediates were obtained commercially (fromBionet): 1-[3-phenyl-benzo[c]isoxazol-5-yl]-ethanone;1-[3-(4-fluoro-phenyl)-benzo[c]isoxazol-5-yl]-ethanone;1-[3-(4-chloro-phenyl)-benzo[c]isoxazol-5-yl]-ethanone;3-dimethylamino-1-(3-phenyl-benzo[c]isoxazol-5-yl)-propenone;3-dimethylamino-1-[3-(4-fluoro-phenyl)-benzo[c]isoxazol-5-yl]-propenone;3-dimethylamino-1-[3-(4-chloro-phenyl)-benzo[c]isoxazol-5-yl]-propenone;and 1-(4-nitro-phenyl)-3-dimethylamino-propenone.

Example 2Phenyl-[4-(3-phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-yl]-amine(Compound I-A1)

3-Dimethylamino-1-(5-methyl-3-methylsulfanyl-1-phenyl-1H-pyrazol-4-yl)-propenone(30 mg, 0.1 mmol) and N-phenylguanidine (15 mg, 1.1 equiv.) wereslurried in acetonitrile (0.5 mL) and heated at 100° C. for 24 hours.The mixture was diluted with methanol (2 mL) and heated briefly andcooled. The resulting solid was filtered and washed with methanol (1mL). The solid was dried under reduced pressure to afford the titlecompound. The compound was characterized by LC/MS and HPLC.

Example 3(4-Fluoro-phenyl)-[4-(3-phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-yl]-amine(Compound I-A2)

Compound I-A2 was prepared according to the procedure described above inExample 2 except that N-phenylguanidine was replaced byN-(4-fluoro-phenyl)-guanidine.

Example 4(6-Chloro-pyridin-3-yl)-[4-(3-phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-yl]-amine(Compound I-A3)

Compound I-A3 was prepared according to the procedure described above inExample 2 except that N-phenylguanidine was replaced byN-(6-chloro-pyridin-3-yl)-guanidine.

Example 5(3-Chloro-phenyl)-[4-(3-phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-yl]-amine(Compound I-A4)

Compound I-A4 was prepared according to the procedure described above inExample 2 except that N-phenylguanidine was replaced byN-(3-chloro-phenyl)-guanidine.

Example 6 4-[4-(3-Phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-ylamino]-benzenesulfonamide (CompoundI-A19)

Compound I-A19 was prepared according to the procedure described abovein Example 2 except that N-phenylguanidine was replaced by4-guanidino-benzenesulfonamide.

Example 7N-{4-[3-(4-Chorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-acetamide(1-A22)

Step A.4-[3-(4-Chlorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine.

To a mixture of sodium pellets (14 mg, 0.609 mmol) in methanol (1 mL) atroom temperature, was added guanidine hydrochloride (10 mg, 0.105 mmol)and commercially available1-[3-(4-chlorophenyl)-benzo[c]isoxazole-5-yl]-3-dimethylamino-propenone(50 mg, 0.153 mmol). The reaction mixture was heated at 80° C. for 18hours. The mixture was cooled to room temperature and diluted with water(6 mL). The granular precipitate was filtered, dissolved indichloromethane, then dried over magnesium sulfate. Purification bysilica gel chromatography (4:1 ethyl acetate/hexane) gave4-[3-(4-chlorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine as ayellow solid (35 mg, 98% yield). ¹H NMR (500 MHz, d₆-DMSO) δ 8.68 (s,1H), 8.35 (d, 1H), 8.25-8.19 (m, 3H), 7,82-7.80 (m, 1H), 7.78-7.72 (m,2H), 7.4 (d, 1H), 6.79 (s, 1H) ppm. LC-MS (ES+) m/e=323.04 (M+H).

Step B.N-{4-[3-(4-Chorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-acetamide.

To a suspension of4-[3-(4-chlorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine intoluene (1.5 mL) at room temperature, was added acetic anhydride (0.5mL). The mixture was heated at 100° C. for 3 hours. The reaction mixturewas diluted with water (6 mL) and the precipitate filtered then washedwith toluene (2×6 mL). Purification was achieved by silica gelchromatography (4:1 ethyl acetate/hexane then 2%methanol/dichloromethane), followed by a 5% aqueous sodium bicarbonatewash (1×50 mL) to give the title compound as a yellow solid (12 mg, 30%yield). ¹H NMR (500 MHz, d₆-DMSO) δ 10.62 (s, 1H), 8.85 (s, 1H), 8.75(d, 1H), 8.31 (d, 1H), 8.25 (d, 2H), 8.02 (d, 1H), 7.85 (d, 1H), 7.75(d, 2H), 2.3 (s, 3H) ppm. LC-MS (ES+) m/e=365.13 (M+H).

Example 8{4-[3-(4-Chlorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-methylamine(I-A23)

This compound was prepared in an analogous manner to that described inExample 2 using 1-methylguanidine hydrochloride to yield the titlecompound as a yellow solid (30 mg, 98% yield). ¹H NMR (500 MHz, d₆-DMSO)δ 8.7 (s, 1H) 8.41 (s, 1H), 8.31-8.2 (m, 3H), 7.82 (d, 1H), 7.72 (d,2H), 7.38 (d, 1H), 7.25-7.2 (m, 1H), 2.95-2.85 (m, 3H) ppm. LC-MS (ES+)m/e=337.04 (M+H).

Example 93-(4-Chlorophenyl)-5-(2-morpholin-4-yl-pyrimidin-4-yl)-benzo[c]isoxazole(I-A24)

This compound was prepared according to the procedure described inExample 13, Step E, except using morpholinoformamidine hydrobromide toyield3-(4-chlorophenyl)-5-(2-morpholin-4-yl-pyrimidin-4-yl)-benzo[c]isoxazoleas a yellow solid (30 mg, 98% yield). ¹H NMR (500 MHz, d₆-DMSO) δ 8.7(S, 1H), 8.5 (d, 1H), 8.3-8.22 (m, 3H), 7.82 (s, 1H), 7.75 (d, 2H), 7.55(d, 1H), 3.85-3.8 (m, 4H), 3.75-3.68 (m, 4H) ppm. LC-MS (ES+) m/e=393.13(M+H).

Example 104-[3-(4-Piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl]pyrimidin-2-ylamine(I-A32)

Step A.5-(2-Methyl-[1,3]dioxolan-2-yl)-3-(4-piperidin-1-yl-phenyl)benzo[c]isoxazole

This compound was prepared in a manner analogous to that described inExample 13, Step B except starting with piperidine and a reactionduration of 2.5 h, giving the title compound, after purification, as abright yellow solid (174 mg, 69% yield). ¹H NMR (500 MHz, CDCl₃) δ8.02-7.81 (m, 3H), 7.53 (d, J=9.25 Hz, 1H), 7.10-6.92 (m, 2H), 4.15-3.96(m, 2H), 3.94-3.71 (m, 2H), 3.47-3.23 (m, 4H), 1.83-1.60 (m, 9H). LC-MS(ES+) m/e=365.19 (M+H).

Step B. 1-[3-(4-Piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl)ethanone

This compound was prepared in a manner analogous to that described inExperiment 17, Step C giving the title compound as an orange oil (42.6mg, 97% yield). ¹H NMR (500 MHz, CDCl₃) δ 8.57-8.47 (m, 1H), 8.01-7.91(m, 2H), 7.88 (dd, J=1.5, 9.4 Hz, 1H), 7.55 (dd, 0.85, 9.4 Hz, 1H),7.08-6.94 (m, 2H), 3.46-3.30 (m, 4H), 2.66 (s, 3H), 1.82-1.59 (m, 6H).LC-MS (ES+) m/e=321.1 (M+H).

Step C. 4-[3-(4-Piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl]pyrimidin-2-ylamine (I-A32)

This compound was prepared in a manner analogous to that described inExperiment 17, Steps D & E giving the title compound as an orange solid(30 mg, 70% yield from1-[3-(4-piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl)ethanone). ¹H NMR(500 MHz, CDCl₃) δ 8.58 (s, 1H), 8.38 (d, J=5.25 Hz, 1H), 8.06-7.85 (m,3H), 7.62 (d, J=9.4 Hz, 1H), 7.16-6.92 (m, 3H), 5.19-4.91 (br s, 2H),3.45-3.25 (m, 4H), 1.82-1.61 (m, 6H). HPLC (cyano column) 14.26 min.LC-MS (ES+) m/e=372.2 (M+H).

Example 114-[3-(3-Piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine(I-A33)

Step A.3-(3-Bromophenyl)-5-(2-methyl-[1,3]dioxolan-2-yl)benzo[c]isoxazole

To a solution of KOH (58 g, 1.03 mol) in MeOH (200 mL) at roomtemperature was added a solution of2-methyl-2-(4-nitro-phenyl)-[1,3]dioxolane (10.7 g, 0.051 mol) and3-bromophenylacetonitrile (11.34 g, 0.058 mol) in MeOH (100 mL). Themixture was stirred at room temperature under a stream of nitrogen for 4days. The product was isolated according to the procedure given inExample 15 Step A (8.5 g, 46% yield). ¹H NMR (500 MHz, CDCl₃) δ8.19-8.15 (m, 1H), 7.97 (d, 4.0 Hz, 1H), 7.90 (s, 1H), 7.67-7.59 (m,2H), 7.50-7.40 (m, 2H), 4.15-4.03 (m, 2H), 1.71 (s, 3H). LC-MS ES+)m/e=361.96 (M+H).

Step B.3-Dimethylamino-1-[3-(3-piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl]-propanone

This was prepared according to the procedure described in Example 13 togive the title compound as a brown solid (141 mg, 48% yield from3-(3-bromophenyl)-5-(2-methyl-[1,3]dioxolan-2-yl)benzo[c]isoxazole). ¹HNMR (500 MHz, DMSO-d₆) δ 8.50 (s, 1H), 8.09-7.89 (m, 1H), 7.89-7.64 (m,2H), 7.63-7.45 (m, 3H), 7.42-7.13 (m, 1H), 6.01 (d, J=12.2 Hz, 1H),3.51-3.27 (m, 4H), 3.26-3.07 (m, 3H), 3.06-2.80 (m, 3H), 1.84-1.42 (m,6H). LC-MS ES+) m/e=371.31 (M+H). HPLC (cyano column) 14.13 minutes.

Step C.4-[3-(3-Piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine(1-A33)

This compound was prepared in a manner analogous to that described inExperiment 17, Step E.

The title compound was isolated as a yellow/brown solid (97 mg, 69%). ¹HNMR (500 MHz, CDCl₃) δ 8.56 (S, 1H), 8.39 (D, J=5.2 Hz, 1H), 7.97 (dd,J=1.3, 9.4 Hz, 1H), 7.69 (d, 9.5 Hz, 1H), 7.63-7.53 (m, 1H), 7.52-7.37(m, 2H), 7.17-7.02 (m, 2H), 5.16 (br s, 2H), 3.39-3.19 (m, 4H),1.86-1.53 (m, 6H). LC-MS ES+) m/e=361.96 (M+H). HPLC (cyano column)12.01 minutes.

Example 12 4-[4-(4-Nitro-phenyl)-pyrimidin-2-ylamino]-benzenesulfonamide

1-(4-Nitro-phenyl)-3-dimethylamino-propenone (3 mmol) and4-guanidino-benzenesulfonamide (3.3 mmol) in acetonitrile (1 mL) wasrefluxed for 36 hours. The mixture was diluted with methanol (5 mL) andcooled to room temperature. The yellow solid was filtered and washedwith methanol (3 mL) and dried under reduced pressure to afford titlecompound. The compound was characterized by LC/MS and HPLC.

Example 134-[3-(4-Morpholin-4-yl-phenyl)benzo[c]isoxazol-5-yl]pyrimidin-2-yl amine(I-A34)

Step A. 3-(4-Bromo-phenyl)-5-(2-methyl-(1,3]-dioxolan-2-yl)-benzo[cisoxazole

To solution of KOH (28.46 g, 508 mmol) in MeOH (50 mL) at 0-10° C. wasadded a solution of 4-bromophenylacetonitrile (6.32 g, 32.2 mmol) and2-methyl-2-(4-nitro-phenyl)-(1,3]-dioxolane (I) (5.35 g, 25.6 mmol) inMeOH (15 mL). The mixture was stirred at room temperature under nitrogenfor 18 hours giving a thick slurry. Water (100 mL) was added and theprecipitate was filtered, and was washed with water (2×75 mL). The solidwas dissolved in hot CH₂Cl₂, filtered and evaporated to give a brownsolid. Repeated triturations with Et₂O gave the product as a brightorange solid (5.19 g, 56% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.99-7.68(m, 1H), 7.79-7.68 (m, 2H), 7.66-7.54 (m, 1H), 7.52-7.40 (m, 1H),4.17-4.04 (m, 2H), 3.92-3.78 (m, 2H), 1.70 (s, 3H) ppm. LC-MS (ES+)m/e=361.9 (M+H).

Step B.5-(2-Methyl-[1,3′-dioxolan-2-yl)-3-(4-morpholin-4-yl-phenyl)-benzo[c]isoxazole

A flame dried, argon flushed flask was charged with3-(4-bromo-phenyl)-5-(2-methyl-[1,3]-dioxolan-2-yl)-benzo[c]isoxazole(199.6 mg, 0.56 mmol), Pd(OAc)₂ (5 mg, 0.02 mmol), P(tBu)₃ (30 μL of 10%solution in toluene, 0.012 mmol), NaOtBu (78.8 mg, 0.82 mmol) andmorpholine (150 μL, 1.72 mmol) in anhydrous toluene (1 mL). The mixturewas heated at 80° C. under Argon for 3 hours. The solvent wasevaporated, and purification by flash chromatography (SiO₂) elutinginitially with 1:9 EtOAc:hexanes to 3:7 EtOAc:hexanes provided the titlecompound as bright yellow solid (49 mg, 24% yield). ¹H NMR (500 MHz,CDCl₃) δ 7.96 (d, 2H), 7.91 (s, 1H), 7.55 (d, J=9.35 Hz, J=8.9 Hz, 1H),7.47-7.34 (m, 1H), 7.04 (d, J=8.95 Hz, 2H), 4.17-4.01 (m, 2H), 3.95-3.76(m, 6H), 3.31 (t, J=5 Hz, 4H), 1.7 (s, 3H). HPLC (cyano column) 8.61minutes

Step C. 1-[3-(4-Morpholin-4-yl-phenyl)benzo[c]isoxazol-5-yl]ethanone

A solution of5-(2-methyl-[1,3]-dioxolan-2-yl)-3-(4-morpholin-4-yl-phenyl)-benzo[c]isoxazole(37 mg, 0.10 mmol) in formic acid (88% solution, 1.5 mL) was stirred atroom temperature for 70 minutes. The formic acid was removed in vacuo,and the resultant solid was dissolved in CH₂Cl₂, dried over sodiumsulfate, filtered and evaporated to give the product as an orange solid(1.42 g, 87% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 8.51 (s, 1H), 7.99 (d,J=8.9 Hz, 2H), 7.88 (d, J=1.0 Hz, 1H), 7.58 (d, 9.4 Hz, 1H), 3.90 (t,J=4.8 Hz, 4H), 3.35 (t, J=5.0 Hz, 4H), 2.66 (s, 3H). LC-MS (ES+)m/e=323.09 (M+H).

Step D.3-Dimethylamino-1-[3-(4-morpholino-4-yl-phenyl)-benzo[c]isoxazol-5-yl]propenone

A solution of 1-(3-(4-morpholin-4-yl-phenyl)benzo[c]isoxazol-5-yl]ethanone (25 mg, 0.08 mmol) in DMF (2.5 mL) wastreated with DMF-DMA (50 μL, 0.37 mmol) and was heated at 90° C. for 36hours and for 100° C. for a further 18 hours. The solvent was evaporatedto give the crude product as brown oil (35.2 mg) which was used directlyin the next step without purification. LC-MS (ES+) m/e=378.2 (M+H).

Step E.4-[3-(4-Morpholin-4-yl-phenyl)benzo[c]isoxazol-5-yl]pyrimidin-2-yl amine(I-A34)

To a solution of sodium (spheres, 25 mg, 1.08 mmol) in MeOH (0.7 mL) atroom temperature under nitrogen was added guanidine hydrochloride (10mg, 0.105 mmol) and a solution of3-dimethylamino-1-[3-(4-morpholino-4-yl-phenyl)-benzo[c]isoxazol-5-yl]propenone(0.08 mmol) in MeOH (1.5 mL) and the reaction was heated to 90° C. for18 hours. The resulting precipitate was filtered to give the product asan orange solid (25 mg, 84% yield). ¹H NMR

MHz, DMSO-d₆) δ 8.61 (s, 1H), 8.33 (d, 1H), 7.98-8.10 (m, 3H), 7.23-6.98(m, 3H), 3.96-3.75 (m, 4H), 3.43-3.30 (m, 4H), 2.62-2.49 (m, 2H). LC-MS(ES+) m/e=374.18 (M+H), HPLC (cyano column) 9.42 minutes.

Example 14 4-{4-[3-(3,4-Dimethoxy-phenyl)-benzo[cisoxazol-5-yl]-pyrimidin-2-ylamino}-benzenesulfonamide (CompoundI-A35)

A mixture of4-[(4-(4-Nitro-phenyl)-pyrimidin-2-ylamino]-benzenesulfonamide (0.2mmol) and 3,4-dimethoxy-phenylacetonitrile (0.4 mmol) in dimethylsulfoxide (2 mL) was treated with 20% sodium ethoxide in ethanol (0.5mL) at ice bath temperature. The mixture was stirred at room temperaturefor 18 hours and diluted with methanol (2 mL). Solid was collected andredissolved in methanol (3 mL) and heated 10 minutes at 80° C. andcooled to room temperature. The solid was recrystallized twice inmethanol to afford yellow title compound. The compound was characterizedby LC/MS and HPLC.

Example 154-[3-(4-Bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine (1-A36)

Step A. 1-[3-(4-Bromophenyl)-benzo[c]isoxazol-5-yl]-ethanone

A solution of3-(4-bromo-phenyl)-5-(2-methyl-(1,3]-dioxolan-2-yl)-benzo[c]isoxazole(Example 1, Step A) (2.13 g, 5.93 mmol) in formic acid (88% solution, 50ml) was stirred at room temperature for 30 minutes, affording a thickyellow precipitate. The formic acid was removed in vacuo, and theresultant solid was dissolved in CH₂Cl₂, dried over sodium sulfate,filtered and evaporated to give the product as an orange solid (1.42 g,76% yield). ¹H NMR (500 MHz, CDCl₃) δ 8.47 (s, 1H), 8.04-7.85 (m, 3H),7.85-7.71 (m, 2H), 7.72-7.57 (m, 1H), 2.68 (s, 3H). HPLC (cyano column)17.68 minutes

Step B.1-[3-(4-Bromo-phenyl)-benzo[c]isoxazol-5-yl]-3-dimethylamino-propenone

This compound was prepared from[3-(4-bromophenyl)-benzo[c]isoxazol-5-yl]-ethanone in an analogousmanner to Experiment 15, Step D except that the reaction duration was 18hours. The product was isolated as a brown solid and was used in thenext step without purification (1.61 g, 97% yield). ¹H NMR (500 MHz,CDCl₃) d 8.84 (s, 1H), 7.98-7.79 (m, 4H), 7.77-7.67 (m, 2H), 7.66-7.51(m, 1H), 5.67 (d, J=12.2 Hz, 1H), 3.31-2.78 (m, 6H).

Step C. 4-[3-(4-Bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine

This compound was prepared in an analogous manner to4-[3-(4-chlorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine (seeExample 13). Purification was achieved by trituration withdichloromethane to yield 4-[3-(4-bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine as a yellow solid (559 mg, 49%yield). ¹H NMR (500 MHz, d₆-DMSO) δ 8.67 (s, 1H), 8.36 (d, 1H), 8.2-8.13(m, 3H), 7.88 (d, 2H), 7.82 (d, 1H), 7.39 (d, 1H), 6.78 (s, 1H) ppm.LC-MS (ES+) m/e=367 (M+H).

Example 163-[4-(3-Phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-ylamino]-benzenesulfonamide(Compound I-A37)

Compound I-A37 was prepared according to the procedure described abovein Example 2 except that N-phenylguanidine was replaced by3-guanidino-benzenesulfonamide.

Example 17N-(4-{3-[3-(2,5-Dimethoxy-pyrimidin-4-yl)-phenyl]-benzo[c]isoxazol-5-yl}-pyrimidin-2-yl)-acetamide(Compound I-A50)

Step A:N-{4-[3-(3-Bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-acetamide

Compound I-A50 was prepared according to the procedure described asabove in Example 7 step B utilizing4-[3-(3-bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine insteadof 4-[3-(4-Chlorophenyl)-benzo (cisoxazol-5-yl]-pyrimidin-2-ylamine.Material was isolated, by removal of the solvent under reduced pressureand trituration with dichloromethane, as a yellow powder (430 mg, 77%yield). ¹H NMR (500 MHz TFA-d) δ 9.15 (s, 1H), 8.85 (d, 1H), 8.41 (d,1H), 8.38 (s, 1H), 8.32 (d, 1H), 8.17 (d, 1H), 8.05 (d, 1H), 7.94 (d,1H), 7.64 (dd, 1H), 2.67 (s, 3H) in ppm. LC-MS (ES+) m/e=409 (M+H).

Step B:N-(4-{3-[3-(2,5-Dimethoxy-pyrimidin-4-yl)-phenyl]-benzo[c]isoxazol-5-yl}-pyrimidin-2-yl)-acetamide

A flask was charged withN-{4-[3-(3-bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-acetamide(100 mg, 0.272 mmol), cesium carbonate (97.7 mg, 0.328 mmol), and2,5-dimethoxypyrimidine-6-boronic acid (55.0 mg, 0.3 mmol). The flaskwas evacuated and back-filled with nitrogen 5-7 times before adding 5 mLof degassed p-dioxane and 1 mL of degassed DMF. To this stirringsolution/suspension was added, 125 μL of a 10% w/v benzene solution oftri-tertbutylphosphine followed by the addition of Pd₂(dba)₃ (25 mg,0.0272 mmol) slurred in 1 mL of degassed DMF. The reaction was stirredunder nitrogen atmosphere, at 80° C. Reaction was followed by HPLC anddeemed to be complete in 4 hours. The reaction mixture was suctionfiltered hot through a pad of diatomaceous earth and washed theprecipitate with DMF and acetonitrile. The filtrate was reduced to anoil under reduced pressure and the crude material purified via HPLCutilizing acetonitrile/water/TFA as the eluent. The material wasisolated as a bright yellow powder (15 mg, 13% yield). ¹H NMR (500 MHzDMSO-d₆) δ 8.93 (s, 1H), 8.6 (s, 1H), 8.31 (s, 1H), 8.29 (d, 1H), 8.25(d, 1H), 8.07 (d, 1H), 7.87 (d, 1H), 7.81 (d, 1H), 7.77 (m, 1H), 4.02 (2close sing, 6H) in ppm. LC-MS (ES+) m/e=469 (M+H)

Example 18(4-(3-(3-Bromo-phenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-carbamicacid ethyl ester (Compound I-A55)

To a stirring solution of4-[3-(3-bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine (75 mg;0.205 mmol) in 1 mL of p-dioxane and 0.5 mL of DMSO, was added 40 μL(45.6 mg, 0.42 mmol) of ethyl chloroformate followed by 73 μL (54.3 mg,0.42 mmol) of diisopropylethylamine. The reaction was stirred at 50° C.,in a sealed vessel, for 8 hours. The solvents were removed under vacuoand the crude material was purified via HPLC with acetonitrile/water/TFAas the eluent. The material was isolated as a yellow powder (30 mg, 32%yield). ¹H NMR (500 MHz, DMSO-d₆) δ 10.5 (br s, 1H), 8.9 (s, 1H), 8.75(d, 1H), 8.35 (m, 3H), 8.3(s, 1H), 8.0 (d, 1H), 7.89 (t, 2H), 7.65 (t,1H), 4.2 (q, 2H), 1.26 (t, 3H) in ppm. LC-MS (ES+) m/e=439/441 (M+H)

Example 19 Thiophene-2-carboxylic acid{4-(3-(3-bromo-phenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-amide(Compound I-A 56)

4-[3-(3-bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine (100 mg;0.272 mmol) was dissolved in 3 mL of a mixture (2:1) of dry DMF/THF andstirred under a nitrogen atmosphere at ambient temperature. Sodiumhydride (15 mg, 0.375 mmol, 60% oil dispersion) was added To thereaction and stirred for 30 minutes. Thiophenecarbonylchloride (32 μL;43.7 mg; 0.299 mmol) in 500 μL of dry DMF was added dropwise over 2minutes and the reaction was stirred for 18 hours at ambienttemperature. Workup was affected by removing the solvents under reducedpressure and the resulting residue was triturated with methyltertbutylether. The crude solid was purified via silica column chromatographywith 5% ethanol in methylenechloride to yield 32 mg of a tan powder; 24%yield. ¹H NMR (500 MHz, DMSO-d₆) δ 11.2 (s, 1H), 8.92 (s, 1H), 8.88 (d,1H), 8.39 (d, 1H), 8.35 (s, 1H), 8.3 (d, 1H), 8.23 (d, 1H), 8.15 (d,1H), 7.95 (d, 1H), 7.9 (d, 1H), 7.87 (d, 1H), 7.65 (t, 1H), 7.24 (t, 1H)in ppm. LC-MS (ES+) m/e=477/479 (M+H).

Biological Methods

IC₅₀ Determination for the Inhibition of GSK-3

Compounds were screened for their ability to inhibit GSK-3β (Amino Acids1-420) activity using a standard coupled enzyme system (Fox et al.(1998) Protein Sci. 7, 2249). Reactions were carried out in a solutioncontaining 100 mM HEPES (pH 7.5), 10 mM MgCl₂, 25 mM NaCl, 300 μM NADH,1 mM DTT and 1.5% DMSO. Final substrate concentrations in the assay were10 μM ATP (Sigma Chemicals, St Louis, Mo.) and 300 μM peptide(HSSPHQS(PO₃H₂) EDEEE, American Peptide, Sunnyvale, Calif.). Reactionswere carried out at 30° C. and 60 nM GSK-3β. Final concentrations of thecomponents of the coupled enzyme system were 2.5 mM phosphoenolpyruvate,300 μM NADH, 30 μg/ml pyruvate kinase and 10 μg/ml lactatedehydrogenase.

An assay stock buffer solution was prepared containing all of thereagents listed above with the exception of ATP and the test compound ofinterest. 59 μl of the test reaction was placed in a 96 well 1/2diameter plate (Corning, Corning, N.Y.) then treated with 1 μl of a 2 mMDMSO stock containing the test compound (final compound concentration 30μM). The plate was incubated for about 10 minutes at 30° C. then thereaction initiated by addition of 7 μl of ATP (final concentration 10μM). Rates of reaction were obtained using a Molecular DevicesSpectramax plate reader (Sunnyvale, Calif.) over a 5 minute read time at30° C. Compounds showing greater than 50% inhibition versus standardwells containing DMSO, but no compound, were titrated and IC₅₀ valueswere determined using a similar protocol in standard 96 well plates withthe assay scaled to a final volume of 200 μl.

In the GSK-3 inhibition assay described above, many of the compounds ofthis invention that were tested were found to provide an IC₅₀ valuebelow one micromolar.

K_(i) Determination for the Inhibition of GSK-3

Compounds were screened for their ability to inhibit GSK-3β (Amino Acids1-420) activity using a standard coupled enzyme system (Fox et al.(1998) Protein Sci. 7, 2249). Reactions were carried out in a solutioncontaining 100 mM HEPES (pH 7.5), 10 mM MgCl₂, 25 mM NaCl, 300 μM NADH,1 mM DTT and 1.5% DMSO. Final substrate concentrations in the assay were20 μM ATP (Sigma Chemicals, St Louis, Mo.) and 300 μM peptide(HSSPHQS(PO₃H₂) EDEEE, American Peptide, Sunnyvale, Calif.). Reactionswere carried out at 30° C. and 20 nM GSK-3β. Final concentrations of thecomponents of the coupled enzyme system were 2.5 mM phosphoenolpyruvate,300 μM NADH, 30 μg/ml pyruvate kinase and 10 μg/ml lactatedehydrogenase.

An assay stock buffer solution was prepared containing all of thereagents listed above with the exception of ATP and the test compound ofinterest. The assay stock buffer solution (175 μl) was incubated in a 96well plate with 5 μl of the test compound of interest at finalconcentrations spanning 0.002 μM to 30 μM at 30° C. for 10 minutes.Typically, a 12 point titration was conducted by preparing serialdilutions (from 10 mM compound stocks) with DMSO of the test compoundsin daughter plates. The reaction was initiated by the addition of 20 μlof ATP (final concentration 20 μM). Rates of reaction were obtainedusing a Molecular Devices Spectramax plate reader (Sunnyvale, Calif.)over 10 min at 30° C. The K_(i) values were determined from the ratedata as a function of inhibitor concentration.

In the GSK-3 inhibition assay described above, many of the compounds ofthis invention that were tested were found to provide a K_(i) valuebelow one micromolar.

JAK Inhibition Assay

Compound inhibition of JAK were assayed by the method described by G. R.Brown, et al, Bioorg. Med. Chem. Lett. 2000, vol. 10, pp 575-579 in thefollowing manner. Into Maxisorb plates, previously coated at 4° C. withPoly (Glu, Ala, Tyr) 6:3:1 then washed with phosphate buffered saline0.05% and Tween (PBST), was added 2 μM ATP, 5 mM MgCl₂, and a solutionof compound in DMSO. The reaction was started with JAK enzyme and theplates incubated for 60 minutes at 30° C. The plates were then washedwith PBST, 100 μL HRP-Conjugated 4G10 antibody was added, and the plateincubated for 90 minutes at 30° C. The plate was again washed with PBST,100 μL TMB solution is added, and the plates were incubated for another30 minutes at 30° C. Sulfuric acid (100 μL of 1M) was added to stop thereaction and the plate is read at 450 nm to obtain the optical densitiesfor analysis to determine IC₅₀ values.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments which utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments which have been represented by way of example.

1-10. (canceled)
 11. A composition comprising a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: A-B is N—O or O—N; Ar is an optionally substituted C₅₋₁₀ aryl group; T is a C₁₋₄ alkylidene chain wherein one or two methylene units of T are optionally and independently replaced by O, NR, S, C(O), C(O)NR, NRC(O)NR, SO₂, SO₂NR, NRSO₂, NRSO₂NR, CO₂, OC(O), NRCO₂, or OC(O)NR; n is zero or one; R¹ is hydrogen or an optionally substituted group selected from C₁₋₁₀ aliphatic. C₅₋₁₀ aryl, C₆₋₁₂ aralkyl, C₃₋₁₀ heterocyclyl, or C₄₋₁₂ heterocyclylalkyl; each R² is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆ aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); each R³ is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆ aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); and each R is independently selected from hydrogen, a C₁₋₈ aliphatic group, or two R on the same nitrogen are taken together with the nitrogen to form a 4-8 membered heterocyclic ring having 1-3 heteroatoms selected from nitrogen, oxygen or sulfur, and a pharmaceutically acceptable carrier, adjuvant or vehicle, additionally comprising an additional therapeutic agent selected from an a chemotherapeutic or anti-proliferative agent, a treatment for Alzheimer's Disease, a treatment for Parkinson's Disease, an agent for treating Multiple Sclerosis (MS), a treatment for asthma, an anti-inflammatory agent, an immunomodulatory or immunosuppressive agent, a neurotrophic factor, an agent for treating cardiovascular disease, an agent for treating liver disease, an agent for treating a blood disorder, or an agent for treating an immunodeficiency disorder.
 12. (canceled)
 13. A method of treating or lessening the severity of a GSK-3- or JAK-mediated disease or condition in a patient, comprising the step of administering to said patient: a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: A-B is N-0 or O—N; Ar is an optionally substituted C₅₋₁₀ aryl group; T is a C₁₋₄ alkylidene chain wherein one or two methylene units of T are optionally and independently replaced by O, NR, S, C(O), C(O)NR, NRC(O)NR, SO₂, SO₂NR, NRSO₂, NRSO₂NR, CO₂, OC(O), NRCO₂, or OC(O)NR; n is zero or one; R¹ is hydrogen or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₅₋₁₀ aryl, C₆₋₁₂ aralkyl, C ₃₋₁₀ heterocyclyl, or C₄₋₁₂ heterocyclylalkyl; each R² is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); each R³ is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic), and each R is independently selected from hydrogen, a C₁₋₈ aliphatic group, or two R on the same nitrogen are taken together with the nitrogen to form a 4-8 membered heterocyclic ring having 1-3 heteroatoms selected from nitrogen, oxygen or sulfur.
 14. The method according to claim 13, wherein said GSK-3-mediated disease is selected from diabetes, Alzheimer's disease, Huntington's disease, Parkinson's, AIDS-associated dementia, amyotrophic lateral sclerosis (AML), multiple sclerosis (MS), schizophrenia, cardiomycete hypertrophy, ischemia/reperfusion and baldness.
 15. The method according to claim 13, wherein said JAK-mediated disease is selected from an immune response, an autoimmune disease, a neurodegenerative disease, or a solid or hematologic malignancy.
 16. (canceled)
 17. The method according to claim 13, comprising the additional step of administering to said patient an additional therapeutic agent selected from a chemotherapeutic or anti-proliferatic agent, a treatment for Alzheimer's Disease, a treatment for Parkinson's Disease, an agent for treating Multiple Sclerosis (MS), a treatment for asthma, an agent for treating schizophrenia, an anti-inflammatory agent, an immunomodulatory or immunosuppressive agent, a neurotrophic factor, an agent for treating cardiovascular disease, an agent for treating liver disease, an agent for treating a blood disorder, or an agent for treating an immunodeficiency disorder, wherein: said additional therapeutic agent is appropriate for the disease being treated; and said additional therapeutic agent is administered together with said composition as a single dosage form or separately from said composition as part of a multiple dosage form.
 18. A method of inhibiting the production of hyperphosphorylated Tau protein in a patient in need thereof, which method comprises administering to the patient a therapeutically effective amount of a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: A-B is N—O or O—N; Ar is an optionally substituted C₅₋₁₀ aryl group; T is a C₁₋₄ alkylidene chain wherein one or two methylene units of T are optionally and independently replaced by O, NR, S, C(O), C(O)NR, NRC(O)NR, SO₂, SO₂NR, NRSO₂, NRSO₂NR, CO₂, OC(O), NRCO₂, or OC(O)NR; n is zero or one; R¹ is hydrogen or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₅₋₁₀ aryl, C₆₋₁₂ aralkyl, C₃₋₁₀ heterocyclyl, or C₄₋₁₂ heterocyclylalkyl; each R² is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); each R³ is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆ aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); and each R is independently selected from hydrogen, a C₁₋₈ aliphatic group, or two R on the same nitrogen are taken together with the nitrogen to form a 4-8 membered heterocyclic ring having 1-3 heteroatoms selected from nitrogen, oxygen or sulfur.
 19. A method of inhibiting the phosphorylation of β-catenin in a patient in need thereof, which method comprises administering to the patient a therapeutically effective amount of a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: A-B is N—O or O—N; Ar is an optionally substituted C₅₋₁₀ aryl group: T is a C₁₋₄ alkylidene chain wherein one or two methylene units of T are optionally and independently replaced by O, NR, S, C(O), C(O)NR, NRC(O)NR, SO₂, SO₂NR, NRSO₂, NRSO₂NR, CO₂, OC(O), NRCO₂, or OC(O)NR; n is zero or one: R¹ is hydrogen or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₅₋₁₀ aryl, C₆₋₁₂ aralkyl, C₃₋₁₀heterocyclyl, or C₄₋₁₂ heterocyclylalkyl; each R² is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); each R³ is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆ aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); and each R is independently selected from hydrogen, a C₁₋₈ aliphatic group, or two R on the same nitrogen are taken together with the nitrogen to form a 4-8 membered heterocyclic ring having 1-3 heteroatoms selected from nitrogen, oxygen or sulfur.
 20. A composition for coating an implantable device comprising a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: A-B is N—O or O—N; Ar is an optionally substituted C₅₋₁₀ aryl group; T is a C₁₋₄ alkylidene chain wherein one or two methylene units of T are optionally and independently replaced by O, NR, S, C(O), C(O)NR, NRC(O)NR, SO₂, SO₂NR, NRSO₂, NRSO₂NR, CO₂, OC(O), NRCO₂, or OC(O)NR; n is zero or one; R¹ is hydrogen or an optionally substituted group selected from C₁₋₁₀ aliphatic, C₅₋₁₀ aryl, C₆₋₁₂ aralkyl, C₃₋₁₀ heterocyclyl, or C₄₋₁₂ heterocyclylalkyl; each R² is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); each R³ is independently selected from R, halo, CN, OR, N(R)₂, SR, C(═O)R, CO₂R, CONR₂, NRC(═O)R, NRCO₂(C₁₋₆aliphatic), OC(═O)R, SO₂R, S(═O)R, SO₂NR₂, or NRSO₂(C₁₋₆ aliphatic); and each R is independently selected from hydrogen, a C₁₋₈ aliphatic group, or two R on the same nitrogen are taken together with the nitrogen to form a 4-8 membered heterocyclic ring having 1-3 heteroatoms selected from nitrogen, oxygen or sulfur, and a carrier suitable for coating said implantable device.
 21. An implantable device coated with a composition according to claim
 20. 